This invention relates to a process for pretreating a sample for endotoxin measurement, and a pretreating solution used for such a process so as to prevent influences of inhibitors and/or substances showing false positives contained in the sample (hereinafter referred to as "inhibitors, etc.") in the measurement of endotoxin concentration in the sample.
Endotoxin concentration in a sample is measured by detecting an activity of an enzyme (protease, etc.) or the gelation reaction which is caused by the reaction between a horseshoe crab hemocyte lysate (hereinafter referred to as "AL solution") and endotoxins.
In such a measurement there is a fear of presence of inhibitors, etc. which influence the activation of enzymes (protease, etc.) and the gelation reaction.
Endotoxins are lipopolysaccharides (LPS) present mainly in cell walls of Gram-negative bacteria and are known as strong pyrogens. Therefore, it is important to detect endotoxins in medical devices and drugs for injection, and the like. The process for determining endotoxins is described in the Pharmacopoeia of Japan and the United States. Further, endotoxins are considered as a main cause of shock in Gram-negative bacterium infections. In clinical diagnosis, the endotoxin measurement in plasma is used for diagnosis of Gram-negative bacterium infections, judgement of therapeutic effects and recuperation of Gram-negative bacterium infections, early diagnosis of endotoxin shock, etc.
On the other hand, the AL solution has a property of being activated by endotoxins and causing activation of enzymes (protease, etc.) and the gelation reaction. By applying this property, simple and non-expensive endotoxin detection methods are widely used in the fields of medical science, pharmacology and microbiology. Examples of such methods are a method for calorimetrically measuring the degree of activation of enzymes (protease, etc.), a method of applying a gelation reaction, i.e. so-called Limulus test (hereinafter referred to as "Limulus test, etc."), etc.
But, in order to measure endotoxins in plasma, it is necessary to conduct pretreatment of the plasma to be tested by some method so as to prevent influences of the inhibitors, etc. contained in the plasma. Methods of pretreatment of plasma now employed for such a purpose are a perchloric acid treatment (H. Tamura et al: Thromb. Res., 27, 51-57, 1982, etc.), a new perchloric acid treatment (K. Inada et al: Microbiol. Immunol., vol. 35(4), 303-314, 1991, etc.), a dilution and heating treatment (M. S. Cooperstock et al: Lancet, 1, 1272, 1975), etc. But, these treatments have various problems therein and cannot be said as desirable treatments.
For example, the perchloric acid treatment comprises adding perchloric acid to plasma, heating at 37.degree. C. for 20 minutes, removing a precipitate of denatured material by a centrifuge at 3000 r.p.m. for 15 minutes, neutralizing a supernatant with sodium hydroxide, and subjecting the thus treated plasma to the measurement. This treatment has various problems in that the procedures are complicated, a part of endotoxins is taken into the precipitate to lower the recovery of endotoxins, etc.
The new perchloric acid treatment comprises adding sodium hydroxide to plasma, heating at 37.degree. C. for 5 minutes, heating at 37.degree. C. for 10 minutes after addition of perchloric acid, dissolving a produced precipitate with sodium hydroxide, adding a Tris buffer solution to the resulting solution so as to adjust the pH, and subjecting the resulting solution to the measurement. This treatment has the same problem as the perchloric acid treatment in that the procedures are complicated.
The dilution and heating treatment comprises diluting plasma with distilled water 10 times, heating at 100.degree. C. for 10 minutes, and subjecting the thus treated solution to the measurement. This treatment is advantageous in that the procedures are simple, but has a problem in that the recovery of endotoxin added to plasma is low very often.
Apart from the above-mentioned treatments, a treatment of addition of Tween 80 (a trade name of a surfactant mfd. by Kao Atlas Co., Ltd.) is reported recently (H. Fukui et al: Record of 8th Endotoxin Clinical Study Meeting, Yo-do-sha, 59-66, 1989). According to this treatment, plasma is diluted and heated, added with Tween 80 so as to make the final concentration 1% w/v, stirred for 5 minutes under ice cooling using a ultrasonic treating machine, and subjected to the measurement. This treatment also has problems in that the procedures are complicated considerably and a special device such as the ultrasonic treating machine is necessary, etc.